Pharmacokinetic and molecular docking studies to pyrimidine drug using Mn3O4 nanoparticles to explore potential anti-Alzheimer activity

Alzheimer disease (AD) is the cause of dementia and accounts for 60–80% cases. Tumor Necrosis Factor-alpha (TNF-α) is a multifunctional cytokine that provides resistance to infections, inflammation, and cancer. It developed as a prospective therapeutic target against multiple autoimmune and inflammatory disorders. Cholinergic insufficiency is linked to Alzheimer's disease, and several cholinesterase inhibitors have been created to treat it, including naturally produced inhibitors, synthetic analogs, and hybrids. In the current study, we tried to prepared compounds may also support the discovery and development of novel therapeutic and preventative drugs for Alzheimer's using manganese tetroxide nanoparticles (Mn3O4-NPs) as a catalyst to generate compounds with excellent reaction conditions. The Biginelli synthesis yields 4-(4-cyanophenyl)-6-oxo-2-thioxohexahydropyrimidine-5-carbonitrile when the 4-cyanobenzaldehyde, ethyl cyanoacetate, and thiourea were coupled with Mn3O4-NPs to produce compound 1. This multi-component method is non-toxic, safe, and environmentally friendly. The new approach reduced the amount of chemicals used and preserved time. Compound 1 underwent reactions with methyl iodide, acrylonitrile, chloroacetone, ethyl chloroacetate, and chloroacetic acid/benzaldehyde, each of the synthetized compounds was docked with TNF-α converting enzyme. These compounds may also support the discovery and development of novel therapeutic and preventative drugs for Alzheimer's disease. The majority of the produced compounds demonstrated pharmacokinetic features, making them potentially attractive therapeutic candidates for Alzheimer's disease treatment.


XRD analysis
The XRD pattern of crystalline sample exhibits diffraction peaks for Hausmanite structure (Mn 3 O 4 ) (Fig. 1).The acquired diffraction peak positions are matched with the standard value and are found to be compatible with [(JCPDS 24-0734)] which is related to tetragonal single phase of (Mn 3 O 4 ).The lattice parameters of the tetragonal unit cell were found to be (a = b = 5.76 Å and c = 9.46 Å) which is consistent with prior reports 22 .No other impurity peaks were observed in the XRD pattern, indicating that no other manganese oxide formulations were detected.www.nature.com/scientificreports/

Morphological and elemental analyses
The morphology, particle size, and crystallinity of the Mn 3 O 4 nanoparticles have been investigated using FE-SEM and TEM micrographs.Figure 2a depicts FE-SEM image of Mn 3 O 4 -NP.The particles were found to be highly agglomerated but homogeneous in size, and their shape was either spherical or cubic 23 .The EDX results shown in Fig. 2b highlighted the incidence of solely Mn and O elements, indicating the exceptional clarity of the synthesized sample.Figure 2c illustrates the typical TEM image of manganese oxide nanostructure (Mn 3 O 4 ) 24 .It shows that the sample consisted mainly of cubic particles and a few spherical structures with particle size found to be in the range 15-70 nm.This observation is consistent with the SEM results 25 .The SAED pattern in Fig. 2d illustrates the polycrystalline nature of the Mn 3 O 4 nanoparticles 26 .

Chemistry
Depending on the pyrimidine moiety, novel heterocyclic compounds were synthesized.Two approaches were used to synthesize the starting material 4-(4-cyanophenyl)-6-oxo-2-thioxohexahydropyrimidine-5-carbonitrile (1).The conventional procedure comprises the reaction of 4-cyanobenzaldehyde with thiourea, or ethyl cyanoacetate, in ethanol under reflux conditions along with few drops of triethylamine.In order to obtain compound (1) with optimal reaction conditions, table (1), the three components' reaction in the presence of Mn 3 O 4 nanoparticles (Mn 3 O 4 -NPs) as a catalytic amount in ethanol under reflux was recorded (Fig. 3).The structure 1 reviewed was clarified with the use of infrared (IR), proton, and carbon-13 nuclear magnetic resonance ( 1 H-NMR and 13 C-NMR).An absorption band at 1721 cm −1 ascribed to the C=O group, an absorption band at 2224 cm −1 attributable to the CN group, a C=S band at 1270 cm −1 , and an absorption band across the range 3343-3521 cm −1 recognized to the 2NH groups were all seen in the IR spectra.Due to the low yield of compounds 1, 2 and 5 in traditional method, the reaction was optimized using Mn 3 O 4 -NPs.It became apparent that the finest features were attained when the Mn 3 O 4 -NPs was elaborated in the reaction as shown in Table 1.The tabulated results showed that Mn 3 O 4 -NPs achived best results in time and yield.

Insilco-studies
Analyzing drug-likeness and oral bioavailability of synthetic compounds utilizing Swiss ADME web tools Early in the drug development process, it is critical to assess the pharmacokinetic features of candidate drugs.Drugs   27 .In Fig. 6, each molecule was tested against anti-Alzheimer to see if it met the RO5 criteria, which included the properties specified in Table 2 and Fig. 6.

Molecular docking
Pyrimidine derivatives are widely known for being sources of medications for a variety of human problems and have been utilized since ancient times.For our research, we selected 9 synthetic pyrimidine derivatives occurring substances with exceptional antioxidant capabilities that primarily function by scavenging free radical species.Significant information has been acquired recently that points to an increase in oxidative stress in the brain of people with AD.According to Badrul and Ekramu, this could play a part in the pathophysiology of neuron degeneration and death 28 .Therefore, defense and suppression of oxidative stress might be regarded as a crucial factor in the creation of anti-Alzheimer medications.www.nature.com/scientificreports/co-crystallized ligand was isolated as a standard reference from TNF-α Converting Enzyme (TACE) in complex with IK682 enzyme (PDB code: 2FV5).This investigation was carried out to get deeper insight into the binding mechanisms of the produced compounds into the protein-binding site of the TACE in association with the enzyme IK682.The co-crystallized ligand was re-docked into the active site consuming the same parameters to confirm the existing docking study at the active site.The best-docked pose's root means square deviation (RMSD) was 2.7122 Å, and the energy score was − 9.6854 kcal/mol, thus validating the docking study with M.O.E.software.In addition, the co-crystallized ligand formed 3 hydrogen bonds with GLY 349 (A), GLY 349 (A), and one with GLU 406 (A), LEU 348 (A) and HIS 405 (A).Although interacted by pi-H with ALA 439 (A) and ALA 439 (A), Fig. 7.Each of the compounds was docked with Alzheimer disease-associated target individually.These compounds showed very good binding affinity with Alzheimer-associated target.Compounds 3, 5, 6, 7 and 8 showed prefect docking score energy, as shown in Tables 3 and 4.
Donepezil was re-docked into the active site consuming the same parameters to confirm the existing docking study at the active site.The best-docked pose's root means square deviation (RMSD) was 1.2173 Å, and the energy score was − 8.223 kcal/mol, Compounds 5, 6, 7 and 8 showed prefect docking score energy compared with commercial drug Donepezil.

Materials and methods
High-grade materials were used to synthesize the target compounds.Chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany).Solvents were provided by Sigma-Aldrich Company.The manufactured Mn 3 O 4 nanoparticles were provided by the National Research Centre (NRC) by means of precipitation technique.The nanoparticle nature and crystallinity were evaluated using a high-resolution transmission electron microscope (HR-TEM) [JEM-2100, Tokyo, Japan].The liquefied solution of the particles was drop-casted onto a carboncoated copper grid and air-dried at ambient temperature in advance of being examined under a microscope.X-ray diffraction (XRD) was used to identify the phase of prepared dried powder using Bruker Axs diffractometer model (D8 Advance, Germany) in the range of 2θ = 15°-80°.Melting points were measured without correction using a digital Electrothermal IA 9100 Series instrument from Cole-Parmer (Beacon Road, Stone, Staffordshire, ST15 OSA, UK).A PerkinElmer CHN 2400 was exploited for C, H, and N analyses.IR spectra were recorded from 4000 to 400 cm −1 using FT-IR 460 PLUS utilizing KBr wafers.The 1 H and 13 C-NMR spectra were verified by means of a Bruker 800 MHz, NMR Spectrometer, using DMSO-d 6 as a solvent, and chemical shifts were stated in δ (ppm) at King Abdullah University of Science and Technology (KAUST).The scores obtained after the molecular docking technique were calculated and reported using the Molecular Operating Environment (MOE) program (2022).

Preparation of Mn 3 O 4 -NPs
To prepare a (0.4 M) solution, 10 g of manganese nitrate (Mn(NO 3 ) 2 •4H 2 O-BHD Laborator, Suppliers, 99%) was dissolved in 500 mL of distilled water.A (2 M) solution of NaOH was dripped into the manganese nitrate solution with stirring at room temperature until the pH of the solution reached 10.At this pH value, the solid precipitation appears white and then turns brown after a few seconds.The chemical reaction continued for two hours; the precipitate was kept overnight to simmer down.Brownish precipitate was then washed several times using distilled water to discard the excess of the NaOH followed by filtration and drying at 100 °C for 4 h.The dried clean brown precipitate was kept in a desiccator up to its use.

Characterization
The processed Mn 3 O 4 -NPs prepared by the precipitation technique are designated by a number of approaches.XRD is accomplished by [Bruker D 8 advance diffractometer, Germany] with Cu Kα radiation (λ = 1.5406Å) to study the crystallinity of the material.Microscopic examination such as field emission scanning electron microscope (FE-SEM, model FEJ Quanta 250 Fei, Netherlands) supplemented with Energy Dispersive X-Ray Analysis (EDX) was used to study the size and surface structure of the as-prepared Mn 3 O 4 -NPs.The samples' www.nature.com/scientificreports/surface was coated with a thin layer of gold through a [S150A sputter coater, Edwards, England] under 0.1 Torr, vacuum 1.2 kV voltage and 50 mA current to develop the scanning of samples.In addition, the nanoparticle nature and crystallinity were experienced by high resolution transmission electron microscope (HR-TEM, Joel model JEM-2100, operating voltage 200 kV, Japan).Aqueous dispersion of the particles was drop-casted onto a copper grid coated with carbon before being air dried at room temperature to be microscopically examined.

Computer prediction of biological activity of synthetic compounds
Based on its structural formula, a computer tool such as the PASS and Swiss ADME web resources may be used to assess the expected profile of biological activity of a drug-like organic molecule whose molecular mass spans from 50 to 1250 Da.The assessment is established on a review of the structure-activity interactions for a sizable training set comprising chemical probes, drug substances, drug candidates at different phases of preclinical and clinical development, pharmaceutical agents, and compounds for which specific toxicity data is available.

Drug-likeness and oral bioavailability analysis of synthetic compounds using Swiss ADME web resources
Early in the drug development progression, it is crucial to analyze the pharmacokinetic characteristics of candidate medications.To represent the excretion of drugs, the total clearance model and renal OCT2 substrate were utilized 27,29 .

Docking study
The software of the molecular operating environment was used to accomplish the molecular modeling for the highest active compounds, which was outlined using ChemDraw Ultra 12.0, as confirmed by Cousins (2011)  and Cheema et al. (2024) 27,30,31 .
For the modeling of the target protein, the identity of appropriate homologous template structures was accomplished by means of automated homology modeling pipeline SWISS-MODEL (achieved by Swiss Institute of Bioinformatics).In place of improvement of the outcomes, London DG force and force field energy were used.All feasible minimizations were made in anticipation of a root mean square deviation (RMSD) gradient of 0.1 kcal mol −1 Å −1 via MMFF 94× (Merck molecular force field 94×) and the partial charges were spontaneously deliberated.The ligand binding affinity was evaluated by means of the scoring function; dock function (S, Kcal/ mol) created by the MOE 2022 software.From the protein data bank, the X-ray crystal structure of the enzyme's (PDB ID: 2FV5, resolution: 2.10 Å) was downloaded in the PDB format.For docking investigations, the enzyme was prescribed after the following arrangement: (i) The water was removed from the protein (ii) Hydrogen atoms (H) with their standard geometry, were added to the structure then reconnect the bonds broken and fixing the potential.(iii) For the large site search in the enzyme structure, MOE Alpha Site Finder was used, and dummy atoms were generated from the resulting alpha spheres.(iv) Analyzing the ligand's interaction with the active sites of the amino acids.The active ligands with the highest Docking Score have the most negative values.All docking processes and scoring were recorded based on established criteria.For docking, the Triangle Matcher placement method and the London dG score tool were employed 21,[32][33][34][35][36][37][38][39][40][41] .

Conclusions
Alzheimer's disease (AD), which affects a vast population globally, is defined by the elderly's loss of memory and learning capacity, our findings contribute to the body of knowledge on drugs that may someday have pharmacokinetic properties, making them prospective therapeutic options for Alzheimer's disease treatment.
The Biginelli synthesis of 4-cyanobenzaldehyde, ethyl cyanoacetate, and thiourea in the presence of Mn 3 O 4 nanoparticle provides 4-(4-cyanophenyl)-6-oxo-2thioxohexahydropyrimidine-5-carbonitrile (1), which is a costeffective and environmentally beneficial procedure.This multi-part method is non-toxic and safe.Compounds www.nature.com/scientificreports/were created by the reaction of compound (1) with various reagents.Together, these ground-breaking studies were used to look into synthetic compounds in preliminary ways.Conventional docking studies were utilized to evaluate the best docked ligands, allowing us to discover the compounds' binding mechanisms.The binding energies of the drug-target interactions are important in determining how well the drug binds to the target macromolecule.

Table 1 .
Optimization of the Reaction Conditions.

Table 2 .
Using Lipinski's rule of five (RO5) of anti-Alzheimer and chosen bioactive substances of synthesized compounds as possible protein inhibitors.

Table 3 .
The binding scores of the promising compound.Significant values are in bold.

Table 4 .
The binding scores, RMSD values, ligand, receptor, interactions, distance, interactions with distance and energy for the promising compounds compared to the reference ligand as a reference for Anti-Alzheimer.Significant values are in bold.